Classifications

• • C—CHEMISTRY; METALLURGY
  • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
  • C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
  • C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
  • C12Q1/26—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving oxidoreductase
  • C12Q1/32—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving oxidoreductase involving dehydrogenase
• • Y—GENERAL TAGGING OF NEW TECHNOLOGICAL DEVELOPMENTS; GENERAL TAGGING OF CROSS-SECTIONAL TECHNOLOGIES SPANNING OVER SEVERAL SECTIONS OF THE IPC; TECHNICAL SUBJECTS COVERED BY FORMER USPC CROSS-REFERENCE ART COLLECTIONS [XRACs] AND DIGESTS
  • Y10—TECHNICAL SUBJECTS COVERED BY FORMER USPC
  • Y10S—TECHNICAL SUBJECTS COVERED BY FORMER USPC CROSS-REFERENCE ART COLLECTIONS [XRACs] AND DIGESTS
  • Y10S435/00—Chemistry: molecular biology and microbiology
  • Y10S435/805—Test papers

Definitions

• a nicotinamide-adenine dinucleotidean alkali lactate salt mixture and a process for the production of said indicator are disclosed.
• test means for the determination of the concentration of lactate dehydrogenase in body fluids.
• My test means is useful for the qualitative detection and quantitative determination of lactate dehydrogenase in sera wherein the test means comprises a reagent composition incorporated within a bibulous carrier.
• lactate dehydrogenase The quantitative determination of lactate dehydrogenase is extremely important in the detection of heart diseases, especially heart attacks, in that, following heart attacks, the concentration of lactate dehydrogenase in the blood rises noticeably over its normal concentration. The early detection of such an abnormal rise in lactate dehydrogenase concentration can therefore obviously lead to a more accurate and rapid diagnosis of heart maladies.
• novel diagnostic test indicators for the determination of the concentration of lactate dehydrogenase, hereinafter sometimes referred to as LDH, in sera comprise a bibulous carrier material such as cellulosic paper which contains therein the dried residue resulting from the impregnation thereof with a series of reagent materials.
• the first reagent material is a tetrazolium salt. This material is capable of imparting to the area of the test indicator contacted with serum 21 color of such varying intensity as to be representative of the concentration of the lactate dehydrogenase in the serum which is added to the indicator.
• These dyes are well known in the art and generally have the formula wherein R, R and R individually, are the same or different aryl or substituted aryl radicals and X is an anion such 'as a halide etc.
• Examples of useful salts of this configuration include 2,3,5-Triphenyl-2H-tetrazolium chloride; 2-(piodophenyl)-3-(p-nitrophenyl)-5-phenyl-2H- tetrazolium chloride (INT); nitroblue tetrazolium; blue tetrazolium; and the like.
• These salts are incorporated into my novel indicator in concentrations ranging from about 0.05 part to about 0.35 part, preferably, from about 0.1 part to about 02 part, based on 100 parts of solution used, in a manner'set forth hereinbelow.
• the second reagent material incorporated into my novel indicator comprises a chromatographic effect preventor which is employed in order to prevent the chromatographic movement of the tetrazolium salt over the surface of the bibulous carrier material.
• materials which can be used for this purpose include poly(methacrylic acid), polyacrylic acid, carboxymethyl cellulose, copolymers of maleic acid and methylvinyl ether and the like. These materials are used in amounts ranging from about 0.1 part to about 3.0 parts, preferably from about 0.5 part to about 2.5 parts, based on parts of solution used.
• the third component impregnated into the bibulous strip is an anti-oxidant which is employed in order to prevent premature coloration of the tetrazolium salt component test indicator.
• suitable antioxidants include the alkylated phenols such as 2,6- ditertiary butyl-p-cresol; butylated hydroxytoluene, 4- t-butyl catechol, octadecyl-3,5-di-t-butyl-4-hydroxy hydrocinnamate; alkylidene bisphenols such as 2,2-
• phite trisnonyl phosphite and various other well known anti-oxidants
• quinones including hydroquinone, hydroquinone 'monomethyl ether, mono-tbutylhydroquinone, 2,5-di-t-butyl hydroquinone, toluhydroquinone, 2,5-di-t-amyl hydroquinone and the like.
• I may also use phenothiazine, hydroxybenzophenone, p-dimethylaminonitrosobenzene, thiodipropionic acid etc.
• anti-oxidant materials are used in amounts ranging from about 0.01 part to 2.0 part, preferably from about 0.02 part to 1.0 part based on 100 parts of solution and may be used in conjunction with the tetrazolium salt or before or after deposition thereof.
• the fourth component impregnated into the bibulous support is diaphorase which is used to catalyze the reduction of the tetrazolium salt with NADH.
• This enzyme is well known in the art and should be employed in concentrations ranging from about 0.02 part to 0.2 part by weight and is perferably used from 0.03 part to 0.10 part based on 100 parts of solution used.
• Nicotinamide-adenine-dinucleotide in admixture with an alkali lactate salt such as lithium lactate, sodium lactate, potassium lactate and the like, comprises the fifth critical constituent of my novel indicator.
• an alkali lactate salt such as lithium lactate, sodium lactate, potassium lactate and the like
• the use of NAD is well known in the art and should be employed in concentrations ranging from about 0.01 part to about 0.20 part and is preferably used from 0.015 part to 0.08 part by weight based on 100 parts of solution.
• the lactate salt is employed in amounts ranging from 0.03 part toabout 1.5 parts and is preferably used from 0.02 part to 0.09 part based on 100 parts of solution used.
• a suitable non-ionic wetting agent any of those which are well-known to the skilled artisan being applicable.
• I may utilize the fatty alkanolamides i.e. the alkanolamine reaction products with fatty acids such as lauric acid or stripped coconut fatty acid, suitable alkanolamines being diethanolamine, monoethanolamine, amonisopropanolamine and the like; the ethylene oxide derived materials, i.e.
• alkyl group is octyl, nonyl or higher, long chain fatty alcohols such as tridecyl alcohol, lanolin, lecethin alcohol etc., long chain fatty acids such as tall oil, oleic acid, abietic acid etc., long chain fatty mercaptans, long chain fatty amines, polyoxypropylene glycol, fatty sorbitan ester; sugar csters i.e.
• Concentrations of from about 0.01 part to about 1.0 part of wetting agent per 100 parts of solution are employed, the wetting agents preferably being added with each component if the components are added singly or in admixture with the components if they are added as a complete admixed system.
• the method employed depends primarily on the material which is being employed as the anti-oxidant for the tetrazolium salt. If the anti-oxidant is organic solvent soluble only, the dry bibulous carrier, usually paper, is impregnated with the reagents in a series of dips. 7
• An aqueous solution of the tetrazolium salt and the chromatographic effect preventor is prepared and the bibulous material is contacted therewith at a pH ranging from about 7.5 to about 8.8, preferably about 7.5 to about 7.8.
• the impregnated material is then dried such as in drying tunnel or forced draft oven and the dried, impregnated carrier is then contacted with an or ganic solvent solution of the anti-oxidant.
• the carrier is again dried.
• a buffer solution of diaphorase and, preferably, a carbohydrate stabilizer, at a pH of about 7.0 to about 7.5, preferably about 7.2 to about 7.5 is then prepared and the twice impregnated, twice dried carrier is impregnated for a third time therewith and dried.
• a buffer solution of the NAD and alkali lactate at a pH of about 8.8 is prepared and the treated paper is again impregnated.
• a fourth drying completes the preparation of the test indicator.
• wetting agents etc. are to be incorporated, they are added during any or all of the impregnations to obtain uniform reagent deposits.
• Materials suitable as the carbohydrate stabilizer include maltose and sorbitol as well as water soluble polymeric ethylene oxides both high and low molecular weight, diethylene glycol and the like in concentrations ranging from about 10.0 parts to about 25.0 parts, preferably about 15.0 parts to about 20.0 parts based on 100 parts of solution used.
• all the reagents may be admixed together in the buffer solution the concentrations of each ingredient being as set forth above except that each is based on the same 100 parts of water, and a one dip-one dry cycle can be employed to product the desired test indicator.
• the multi-treated paper is dried and a fourth coating saturation therewith to produce a functional test indidded, composed of 008 part of nicotinamide-adenine cator, the absorptive capabilities of the bibulous mate dinucleotide (NAD), 0.025 part of additional wetting rial being characteristic of materials generally used for nt nd 084 part of lithium lactate in 100 parts of this p p water, pH 8.8.
• NAD bibulous mate dinucleotide
• the cellulosic filter paper of Example 1 is then EXAMPLE 1 dipped in this solution and the saturated paper dried Commercially available Cellulose P p 200 under vacuum at room temperature in the dark.
• Each indicator produced contains diat room temperature (26C.) in the dark and dipped in aphorase and NAD as set forth in Example 1.
• the cona second solution containing 1.0 part dilaurylthiodiprocentration, in parts per 100 parts of water or solvent, pionate anti-oxidant and 100 parts of hexane. This of each component is indicated therewith.
• In each inpaper is again dried and dipped in a third solution constance, an excellent test indicator is produced.
• a diagnostic test indicator for the determination of the concentration of lactate dehydrogenase in sera comprising a bibulous material which contains therein the dried residue resulting from the impregnation thereof with l. a tetrazolium salt,
• a diagnostic test indicator according to claim 1 wherein said (1) is 2-(p-iodophenyl)-3-(pnitrophenyl)-5-phenyl-2H-tetrazolium chloride.
• a process for the preparation of the diagnostic test indicator of claim 1 which comprises impregnating a bibulous material with an aqueous solution of said l) and (2), thereafter drying the thus impregnated material, impregnating the thus impregnated material with an organic solvent solution of said (3), thereafter drying the thus twice impregnated material, impregnating the this twice impregnated material with a solution of said (4), thereafter drying the thrice impregnated "material, and then impregnating the thus thrice impregnated material with (5) and drying the thus four times impregnated material.
• a process according to claim 9 wherein said (1) is 2-(p-iodophenyl)-3-(p-nitrophenyl)-5-phenyl 2H- tetrazolium chloride.
• a process according to claim 9 wherein said alkali lactate salt is lithium lactate.

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• General Engineering & Computer Science (AREA)
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• Genetics & Genomics (AREA)
• Investigating Or Analysing Biological Materials (AREA)
• Measuring Or Testing Involving Enzymes Or Micro-Organisms (AREA)

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Cited By (17)

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Citations (1)

• 1973
  • 1973-11-08 US US414035A patent/US3867259A/en not_active Expired - Lifetime
• 1974
  • 1974-10-01 ZA ZA00746459*7A patent/ZA746459B/xx unknown
  • 1974-10-21 AR AR256170A patent/AR202236A1/es active
  • 1974-10-29 BR BR9033/74A patent/BR7409033A/pt unknown
  • 1974-11-04 DE DE19742452283 patent/DE2452283A1/de active Pending
  • 1974-11-06 DK DK578874A patent/DK578874A/da unknown
  • 1974-11-07 IT IT53922/74A patent/IT1023182B/it active
  • 1974-11-07 FR FR7436933A patent/FR2251001B3/fr not_active Expired
  • 1974-11-07 SE SE7413999A patent/SE7413999L/xx unknown
  • 1974-11-07 BE BE150282A patent/BE821937A/xx unknown
  • 1974-11-07 NL NL7414529A patent/NL7414529A/xx unknown
  • 1974-11-08 JP JP49128146A patent/JPS5081194A/ja active Pending
  • 1974-11-08 DD DD182251A patent/DD116314A5/xx unknown

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